The Journal of Neuroscience, January 13, 2010 30(2):739 748 739

Development/Plasticity/Repair

A Large-Scale Chemical Screen for Regulators of the

Arginase 1 Promoter Identifies the Soy Isoflavone Daidzein
as a Clinically Approved Small Molecule That Can
Promote Neuronal Protection or Regeneration via a
cAMP-Independent Pathway
Thong C. Ma,1* Aline Campana,2* Philipp S. Lange,1,8 Hsin-Hwa Lee,1 Kasturi Banerjee,1 J. Barney Bryson,2
Lata Mahishi,1 Shabnam Alam,6 Roman J. Giger,3,6 Stephen Barnes,7 Sidney M. Morris Jr.,4 Dianna E. Willis,5
Jeffrey L. Twiss,5 Marie T. Filbin,2 and Rajiv R. Ratan1
1

Burke-Cornell Medical Research Institute, White Plains, New York 10605, 2Biology Department, Hunter College, New York, New York 10021, 3University
of Michigan School of Medicine, Ann Arbor, Michigan 48109, 4University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, 5Nemours
Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, Delaware 19899, 6University of Rochester, Rochester, New York 14642,
7University of Alabama Birmingham, Birmingham, Alabama 35294, and 8Department of Anesthesiology and Intensive Care Medicine, University of Bonn,
53127 Bonn, Germany

An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been
shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by
myelin proteins. To identify small molecules that capture Arg1s protective and regenerative properties, we screened a hippocampal cell
line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds
containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons
primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by
acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was
effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most
importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for
its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB
phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP
modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood brain barrier, and is
effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.

Introduction
Despite significant focus on neuroprotection, stroke and spinal
cord injury (SCI) remain prevalent causes of disability worldwide
as therapeutics designed to limit cell death have failed to enhance
recovery in humans (Baptiste and Fehlings, 2007; Savitz and

Received Oct. 23, 2009; revised Nov. 19, 2009; accepted Nov. 24, 2009.
This work was supported by National Institutes of Health (NIH) Grants NS040591 to R.R.R. and GM057384, a New
York State Spinal Cord Injury Research Board Center of Research Excellence Grant to R.R.R., M.T.F., and R.J.G.
(CO19772), Adelson Foundation grants to R.R.R. and J.L.T., the Purdue University-University of Alabama at Birmingham Botanicals Center for Age-Related Disease grant from the National Center for Complementary and Alternative
Medicine and the NIH Office of Dietary Supplements (P50 AT00477, C. Weaver, Principal Investigator), and the Wings
for Life Foundation. We thank Dr. Alvaro Estevez and Dr. Marco Domeniconi for their assistance with this manuscript.
*T.C.M. and A.C. contributed equally to this manuscript.
Correspondence should be addressed to Dr. Rajiv R. Ratan, Burke-Cornell Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605. E-mail: rratan@burke.org.
DOI:10.1523/JNEUROSCI.5266-09.2010
Copyright 2010 the authors 0270-6474/10/300739-10$15.00/0

Fisher, 2007; Ratan and Noble, 2009). As such, therapies that

both protect neurons and foster CNS repair may facilitate
progress beyond the limited benefits of currently available agents.
Functional recovery after injury requires many barriers to be surmounted, including inhibitory myelin proteins (Cafferty and
Strittmatter, 2006; Hannila and Filbin, 2008). Indeed, direct neutralization of these proteins has resulted in improved recovery
after injury (Rossignol et al., 2007). Alternatively, neurons can be
induced to ignore inhibitory cues activated by myelin (Cai et
al., 1999).
Paradigms to develop novel neuroprotectants that also target
CNS repair have come from prior elegant studies. Priming of
DRG neurons by conditioning peripheral lesion or with neurotrophic factors allows neurons to resist myelin inhibitors; both
mediate their beneficial effects by increasing cAMP (Filbin,
2003). Excitingly, agents that enhance cAMP levels promote axonal regeneration in injury models (Neumann et al., 2002; Qiu et

740 J. Neurosci., January 13, 2010 30(2):739 748

al., 2002; Lu et al., 2004; Nikulina et al., 2004; Pearse et al., 2004).
These regenerative effects are transcription-dependent and are
mediated by arginase 1 (Arg1), a notable gene target for cAMPmediated transcription. Arg1 catalyzes the conversion of arginine
to urea and ornithine, a rate-limiting step for polyamine synthesis (Lange et al., 2004). Increased polyamines confer resistance of
neurons to myelin inhibition and spermidine promotes optic
nerve regeneration in vivo (Cai et al., 2002; Deng et al., 2009).
Arg1 also protects motor neurons from trophic factor deprivationinduced apoptosis and cortical neurons from excitotoxicity by
reducing arginine critical for nitric oxide (NO) synthesis (Dawson et
al., 1991; Estevez et al., 2006). Collectively, these studies suggest
that increasing arginase activity by elevating cAMP may be a
promising therapeutic approach to promote CNS protection and
regeneration.
An established strategy to increase cAMP in the CNS employs
isoform-selective inhibition of phoshodiesterases (PDE) to suppress cAMP degradation. PDE inhibitors (e.g., rolipram) increase
cAMP and promote regeneration in rodent models of SCI (Lu et
al., 2004; Nikulina et al., 2004; Pearse et al., 2004) and prevent
neuronal loss in stroke models (Kato et al., 1995; Block et al.,
1997). However, their clinical use is limited by disabling nausea
in some patients attributable to increases in cAMP in CNS emesis
centers (Spina, 2008).
To identify drugs capable of protecting neurons and promoting axonal growth following injury, we focused on Arg1 for its
ability to confer some of cAMPs salutary effects (Cai et al., 2002;
Estevez et al., 2006). Indeed, we previously showed that cAMP
induced Arg1 message and protein levels (Cai et al., 2002), and
reasoned that an Arg1 promoter-reporter construct expressed in
neuronal cells could be used to identify novel transcriptional activators of Arg1. As targeting transcription risks inadvertent toxicity, we screened a 2000 compound small molecule library that
included clinically approved drugs and diverse natural compounds with a long record of safety in humans (Heemskerk et al.,
2002).

Materials and Methods

All protocols involving animals were approved by the respective ILACUC
committees of each institution and the NIH guidelines for use and care
for laboratory animals. Animals used for the studies were obtained from
Charles River Laboratories or Harlan.
Arg1 promoter screen. HT22 cells stably transfected with a luciferase
reporter driven by 4.8 kb of DNA upstream of the Arg1 transcription
start site were plated in 96 well culture plates for 24 h (Gray et al., 2005).
Drug stocks (1000) in DMSO (MicroSource Spectrum library) were
diluted in two steps (1:20 first, then 1:50 into final treatment media) to
minimize precipitation, and then added to the plated cells. After incubation for 24 h, luciferase activity was measured with a firefly luciferase
assay (Promega) in an LMaxII luminometer (Molecular Devices). Luminescence readings were then normalized to well protein content measured by BCA protein assay (Pierce). As a secondary screen, HT22 cells
were treated for 24 h with the compounds that increased Arg1 reporter
activity and then protein lysates were collected for Western blot analysis
for Arg1 protein levels.
Neurite outgrowth assay on MAG-expressing CHO cells. The neurite
outgrowth assay was performed on confluent monolayers of control
CHO cells or myelin-associated glycoprotein (MAG)-expressing CHO
cells in individual chambers of 8 well chamber slides (Nunc). Neurons
were isolated from the DRGs or cerebellum of postnatal day 5 (P5)P6
rats or from the cortices of embryonic day 18 (E18) rat embryos and
cultured as previously described (Mukhopadhyay et al., 1994). For in
vitro priming experiments, neurons were plated onto poly-L-lysinecoated culture dishes and incubated with the indicated drugs or vehicle
overnight, trypsinized, and then resuspended in modified DMEM with

Ma et al. Protective Daidzein Overcomes Glial Inhibition

Sato supplement [200 nM progesterone, 224 nM selenium, 4 g/ml insulin, 0.35 mg/ml bovine serum albumin (BSA), 0.4 g/ml L-thyroxine,
0.34 g/ml triiodothyronine]. Then, 1.5 10 4 DRG, 5 10 4 cerebellar,
or 5 10 4 cortical neurons were plated upon the CHO monolayers for
18 48 h, after which the cocultures were fixed with 4% paraformaldehyde (PFA) and permeabilized with ice-cold methanol for 2 min. The
cells were then blocked for 30 min in DMEM containing 10% fetal bovine
serum (FBS) and incubated overnight with a rabbit polyclonal antibody
against III-tubulin (1:1000, Tuj1, Covance) diluted in 5% BSA/PBS.
The cells were then washed three times with PBS and incubated with a
biotinylated donkey anti-mouse IgG antibody (1:500, GE Healthcare) for
30 min at room temperature, washed three times with PBS, incubated
with streptavidin-conjugated Texas Red (1:300, GE Healthcare) for 45
min, again washed three times with PBS, and mounted with Permafluor
(Immunon). Images were captured with an epifluorescence microscope
and analyzed using MetaMorph (Meta Imaging Software, Molecular Devices) image analysis software. The length of the longest neurite was
determined for each neuron for the first 400 600 neurons encountered
when scanning the slide in a systematic manner.
Turning response to focal sources of MAG. L4 6 DRGs from adult male
Sprague Dawley rats were dissociated using collagenase and trypsin,
plated at a low density on poly-L-lysine and laminin-coated surfaces, and
cultured at 37C with 5% CO2 as previously described (Twiss et al., 2000).
At the time of plating, neurons were cultured in the presence or absence
of 80 M 5,6-dichlorobenzimidazole riboside (DRB), which inhibits
RNA polymerase II. After 12 h, cultures were treated with DMSO or
daidzein (10, 20, 30 or 40 M) for an additional 12 h. For localized
application of tropic stimuli to growth cones, 2.5 g of MAG-Fc (R&D
Systems) was covalently coupled to 4.5 m carboxylated polystyrene
microspheres (2.5 10 7 microparticles) per manufacturers instructions
(Polysciences). Fc-coated microspheres were used to test any nonspecific
effects of coated microparticles. Ligand-immobilized microparticles
were added to DRG cultures 24 h after plating. After allowing the particles to settle for 10 min, DRGs were imaged using a Nikon Eclipse TS100
inverted microscope fitted with a QICAM 12-bit Fast1394 camera. Two
hours after addition of the coated microparticles, DRGs were imaged
again. The turning angle relative to the direction of the last 10 m segment of the axon shaft at the beginning of the experiment and the percentage of growth cone collapse were determined.
Western blot analysis. Cerebellar or cortical neurons plated on poly-Llysine or HT22 cells were treated with the indicated drugs, rinsed and
then lysed with either RIPA or NP-40 buffer supplemented with phosphatase and proteinase inhibitors. Lysates were boiled for 5 min in denaturing buffer after which proteins were separated by SDS-PAGE through
a 10% polyacrylamide gel and then transferred to nitrocellulose membranes. Blots were immunolabeled with antibodies against Arg1 (1:5000)
(Esch et al., 1998), phosphorylated CREB (Ser-133, 1:1000, Millipore), or
CREB (1:1000, Millipore) overnight at 4C, washed, and either incubated
at room temperature for 1 h with HRP-conjugated antibodies against
rabbit IgG (1:5000) or with IR-dye-conjugated antibodies against either
rabbit or mouse IgG (1:15000, LI-COR Biosciences) and then rinsed
three times. Blots incubated with HRP-conjugated secondary antibodies
were visualized by chemiluminescence (ECL reagent, GE Healthcare)
followed by exposure to radiology film. These blots were then stripped
and reprobed with antibodies against -actin (1:1000, Sigma). Blots incubated with IR-dyes were imaged with the Odessey IR quantitative
Western blot system (LI-COR Biosciences).
Intrathecal and subcutaneous daidzein administration. Osmotic minipumps (Alzet model 1007D, 0.5 l/h delivery rate) were filled with various concentrations of daidzein dissolved in DMSO or vehicle only
(Sigma) and then incubated at 37C for at least 6 h before implantation.
For intrathecal delivery, P28 P30 Fisher rats were anesthetized with
isoflurane and the spinal cord at L5 was exposed by laminectomy. An
incision was made in the dura mater and the tip of a cannula attached to
the minipump was guided into the subdural space along the surface of the
spinal cord and secured by a suture thread. For subcutaneous delivery,
minipumps were inserted per manufacturers protocols. Briefly, equilibrated osmotic minipumps were implanted subcutaneously through a
midscapular incision in deeply anesthetized rats. The filled minipumps

Ma et al. Protective Daidzein Overcomes Glial Inhibition

were inserted delivery portal first, after which the surgical opening was
closed with wound clips. Animals were killed 1 d after surgery and the
L4-L5 DRG neurons were isolated and plated upon CHO monolayers for
the neurite outgrowth assay as described above.
Optic nerve crush and intraocular and subcutaneous daidzein administration. Fisher rats (250 300 g) were deeply anesthetized with isoflurane
and immobilized in a stereotaxic apparatus. Optic nerves were surgically
exposed and the dural sheath surrounding the optic nerve was carefully
incised longitudinally. The optic nerve was then crushed 2 mm from the
orbit with a #5 jewelers forceps for 10 s. The surgical site was sutured
closed and the preservation of the central retinal artery was verified by
direct fundoscopic examination for signs of ischemic damage. Animals
showing ischemic damage were excluded from the study. Immediately
following the nerve crush, animals were either injected intraocularly with
10 l of the indicated concentration of daidzein or DMSO with a pulledglass pipette using a nano-injector (WPI) or implanted subcutaneously
with an osmotic minipump loaded with daidzein as described above.
Animals were observed for postoperative recovery and were housed individually with ad libitum access to food and water. Two weeks later,
animals were deeply anesthetized with ketamine/xylazine (100 mg/kg
and 10 mg/kg respectively) and transcardially perfused with 200 ml of
heparinized saline (1000 U/L) followed by 300 ml of 4% PFA. The optic
nerve was removed, postfixed overnight in 4% PFA, and cryoprotected in
30% sucrose in Tris-buffered saline (TBS). Additionally, the lens from
each eye was examined for injury. Animals exhibiting lens injuries were
excluded from the study.
Immunohistochemistry and image analysis. Optic nerves were embedded in freezing medium and 20 m serial sections were cut and mounted
onto glass slides. The sections were washed four times with TBS, blocked
with TBS 0.2% Triton X-100 5% normal goat serum for 1 h, and
then incubated overnight at 4C with a sheep antibody against growthassociated protein-43 (GAP-43; 1:100, a generous gift from Dr. L. Benowitz, Harvard School of Medicine, Boston, MA). The sections were
then washed four times with TBS and incubated with a FITC-conjugated
goat antibody against sheep IgG (1:500) for 1 h at room temperature.
Sections were again washed and mounted with an aqueous mounting
medium before imaging. A high resolution image montage was automatically acquired from a Nikon Eclipse 90i fluorescent microscope at 20
magnification and assembled using MetaMorph acquisition software.
The distance traversed by the three longest GAP-43-positive axons relative to the distal edge of the crush injury site was measured from three
sections for each animal.
Neuron survival assays. Spinal motor neurons were purified from
E14.5 rat embryos as previously described. Briefly, ventral spinal cord
tissue was dissected and dissociated, and enriched for motor neurons by
centrifugation through an Optiprep gradient. Motor neurons were further purified by magnetic bead cell separation (Miltenyi Biotec) for cells
expressing P75 NTR receptor, which is expressed in motor neurons at this
developmental age. The resulting cultures were 96% motor neurons as
assessed by Islet 1/2 immunocytochemistry. The cells were plated in 96
well plates in Neurobasal media B27 supplement (Invitrogen) in the
presence or absence of brain-derived neurotrophic factor (BDNF, 1 ng/
ml). Daidzein (20 M) or DMSO was added at the time of plating. Motor
neurons were assayed for viability 24 h later using calcien-AM (Invitrogen) uptake followed by unbiased cell counting using a Flash Cytometer
(Trophos). Cortical neurons were cultured from E17 rat embryos as
previously described. After 1 d in culture, neurons were cotreated with
daidzein and homocysteic acid (HCA, 5 mM), which prevents the uptake
of cystine and causes glutathione depletion leading to apoptosis in embryonic neurons. The cultures were assayed for viability 24 h later with an
MTT assay (Promega) according to the manufacturers protocols.
cAMP measurements. Intracellular cAMP was measured with a competitive colorimetric immunoassay kit according to the manufacturers
protocols (Millipore). In this assay, alkaline phosphatase-conjugated
cAMP competes with cAMP from cell lysates for binding to a cAMPspecific antibody. The conjugated cAMP is then detected using an alkaline phosphatase substrate. Primary cortical neurons [1 day in vitro
(DIV)] were treated overnight with daidzein and then assayed for cAMP.
The alkaline phosphatase activity of each sample was measured in a Spec-

J. Neurosci., January 13, 2010 30(2):739 748 741

traMax spectrophotometer (Molecular Devices) and compared with a

cAMP standard curve for quantitation.
Estrogen promoter activity. Estrogen promoter activity was assessed
using a firefly luciferase reporter gene under the control of three copies of
the vitellogenin estrogen response element (ERE) (Hall and McDonnell,
1999). Naive HT22 cells were transiently transfected with the ERE reporter using Lipofectamine 2000 (Invitrogen) and then treated with 17estradiol with or without fulvestrant, an estrogen receptor antagonist.
After 24 h, the cells were lysed and assayed for luciferase activity as above.
Statistical analysis. All data were expressed as mean SEM and were
plotted using GraphPad Prism software. The statistical tests used for
analysis are indicated in the figure legends. Statistical significance was
taken as p 0.05.

Results
Biological screening of drug library
We generated a pool of stable immortalized hippocampal cell
clones [HT22ap (arginase promoter)] that express a firefly luciferase reporter driven by a large portion of the proximal 5 noncoding region (4.8 kb) of the murine Arg1 gene. To evaluate the
validity of this tool for analyzing arginase transcription in a neuronal context, we first examined the effect of dibutyryl-cAMP
(db-cAMP), a membrane-permeable and nonhydrolyzable form
of the second messenger cAMP, that was previously shown to
induce Arg1 transcription in primary cortical and DRG neurons
(Cai et al., 2002). As expected, we found robust and reproducible
induction of Arg1 mRNA in HT22 cells. This induction of message was accurately reported by our HT22ap line. Induction of
Arg1 by db-cAMP was attributable to increases in cAMP (and not
butyrate) as it effects were mimicked by the PDE4 inhibitor rolipram. Moreover, actinomycin D, a canonical inhibitor of transcription, completely abrogated the cAMP-mediated increase in
Arg1 message and luciferase activity, demonstrating that the cell
line and reporter system were ideal tools to identify compounds
to regulate Arg1 in neurons (data not shown).
To assess the induction of Arg1 in HT22ap line, we screened a
commercially available small molecule library containing 2000
Food and Drug Administration (FDA)-approved drugs, natural
products, and other biologically active compounds (MicroSource Spectrum library) using a luminometer to detect luciferase production (Fig. 1 A, B). From this initial screen, we
identified 40 compounds that induced luciferase activity by at
least twofold, an amount similar to our positive control, 1 mM
db-cAMP (Fig. 1 B). Hits from the initial screen (performed at
10 M) such as daidzein exhibited a concentration-dependent
induction of activity at the Arg1 promoter (Fig. 1C). To confirm
that changes in reporter activity reflected increases in protein, we
performed immunoblot analysis on native HT22 cells using an
antibody specific for Arg1 (Fig. 1 D) (Estevez et al., 2006). Of the
small molecules that increased reporter activity, only 10 increased Arg1 protein levels. Interestingly, many inducers of Arg1
protein were isoflavones or intermediates/precursors to flavanoid biosynthetic pathways (Fig. 1 E). However, not all isoflavones were true hits. For example, daidzein induced Arg1 levels
whereas genistein, which differs from daidzein by only one hydroxyl group, was ineffective (Fig. 1 D, E; lane 13 daidzein, lane
20 genistein). Additionally, while many flavones increased reporter activity, only methoxyvone was positive in the secondary
screen (Fig. 1 D, E; lane 12 4,7-dimethyoxyflavone, lane 31
methoxyvone, lane 32 ginkgetin).
From the initial 40, we selected 12 compounds, three true
hits and nine false positives, to test in a blind screen for the
ability to overcome neurite outgrowth inhibition by MAG.
Using a model previously developed in one of our labs, cerebellar

742 J. Neurosci., January 13, 2010 30(2):739 748

Ma et al. Protective Daidzein Overcomes Glial Inhibition

granule neurons (CGN) were plated upon

a monolayer of control CHO cells or upon
CHO cells expressing MAG (MAG-CHO)
(Mukhopadhyay et al., 1994). Plating
neurons on MAG-CHO cells significantly
inhibits neurite outgrowth. When added
concurrently with neurons at plating, no
effect on MAGs inhibition of neurite outgrowth was seen with any of these 12 compounds (at 5, 10, and 30 M). In contrast,
db-cAMP, a positive control, completely
overcame MAG inhibition as previously
reported (data not shown). Interestingly,
neurotrophins added at plating are also
unable to promote neurite outgrowth on
MAG substrates; however neurons primed
with neurotrophins before plating are
able to overcome MAG inhibition (Cai
et al., 1999). Using this priming protocol, we first plated CGNs on poly-llysine-coated plates in the presence of
the same amounts of each compound
overnight. The treated neurons were then
trypsinized, transferred onto monolayers of control or MAG-expressing CHO
cells, and allowed to grow overnight before fixation and immunolabeling for
III-tubulin. Under these conditions,
only one compound was able to completely overcome MAG inhibition, while
one other promoted modest neurite outgrowth (Fig. 2 A). Importantly, these two
compounds, daidzein and methoxyvone,
were among the three true hits identified
in the Arg1 screen, validating our strategy of using Arg1 induction as a biological marker to screen for molecules that
overcome myelin-associated axonal growth
inhibition.
Daidzein is effective in many types of
neurons and requires arginase activity
to overcome MAG inhibition
As daidzein was the most effective comFigure 1. Biological screen for inducers of Arg1. A drug library containing 2000 FDA-approved drugs, natural products, and
pound in overcoming MAG inhibition, other biologically active small molecules was screened for inducers of Arg1 using a luciferase reporter driven by 4.8 kb of the Arg1
we further characterized daidzein in vitro promoter in HT22 cells. A, Schematic representation of the screening procedure. B, Representative plot of data from one 96 well
to assess its potential as a therapeutic plate. Molecules that induced Arg1 reporter activity by at least twofold were selected as hits. C, Daidzein concentrationresponse
agent (Fig. 2 A). Using increasing concen- curve (M) for activity at the Arg1 promoter. D, Protein lysates from cells treated with indentified compounds were immunoblottrations of daidzein, we found that prim- ted for Arg1 protein levels as a secondary screen. Not all compounds identified in the reporter screen increased Arg1 protein levels.
ing with 20 M was most effective in The lanes are: S, recombinant Arg1 protein; 12, 3,7-dimethyloxyflavone; 13, daidzein; 20, genistein; 31, methoxyvone; and 32,
promoting neurite outgrowth of CGNs on ginkgetin. E, Of the 10 compounds identified and confirmed in the secondary screen, many were flavanoids including daidzein,
MAG-CHO cells (data not shown). This methoxyvone, and derrustone.
dose response for daidzein was reproon MAG CHO cells was completely inhibited by coincubation
duced in three separate laboratories in three different instituwith the antagonist (Fig. 2CG) (Cai et al., 2002). Importantly,
tions, providing internal replication for the initial observations
nor-NOHA alone did not inhibit neurite outgrowth in CGNs
(Fig. 2 H, I and data not shown). At 20 M daidzein, Arg1 protein
plated on control CHO cells (Fig. 2C,G). Together, these data
levels were increased threefold following overnight incubation,
show
that daidzein upregulates Arg1 protein levels and that argiwhich is similar in magnitude to treatment with 2 mM db-cAMP,
nase activity is necessary for overcoming MAG-mediated inhibiwhich we have previously shown to increase Arg1 levels (Fig. 2 B).
tion of axonal outgrowth in CGNs.
As arginase activity is required for cAMP and neurotrophins to
Recent studies have highlighted the cell type-specific roles
overcome MAG inhibition, we used a specific inhibitor of argithat Nogo receptors play in inducing glial inhibition (Mehta et
nase activity, N -hydroxy-nor-L-arginine (nor-NOHA), and
al., 2007; Venkatesh et al., 2007). In contrast, cAMP appears to
found that the daidzein-mediated increase in axonal outgrowth

Ma et al. Protective Daidzein Overcomes Glial Inhibition

J. Neurosci., January 13, 2010 30(2):739 748 743

zein acts in a transcription-dependent

manner to overcome MAG inhibition in
different types of primary neurons.
Daidzein is biologically active in vivo
and crosses the blood brain barrier
In dissociated cultures, neurons have direct access to drugs applied to the culture
medium. To determine whether daidzein
reaches its effectors in vivo, we administered daidzein intrathecally to see whether
local delivery of the drug was sufficient to
prime DRG neurons to overcome MAG
inhibition. Daidzein at 20 or 40 M was
continuously administered through osmotic minipumps for 24 h. The animals
were then killed, DRGs removed, dissociated, and directly plated upon control
CHO or MAG-CHO monolayers. DRG
neurons from animals receiving vehicle
(DMSO) exhibited significant neurite
outgrowth on control CHO cells, whereas
this neurite outgrowth was significantly
blunted on MAG-CHO cells (Fig. 4 A, B).
Most important, DRG neurons from animals receiving 40 M daidzein overcame
inhibition by MAG (Fig. 4C,G) while the
20 M dose had no effect (data not
shown), indicating that daidzein delivered
locally in vivo is biologically active at the
concentrations effective in vitro.
While intrathecal delivery of daidzein
was effective, this route of administration
is inconvenient, invasive, and has potential for serious complications, particularly
Figure 2. Daidzein overcomes MAG inhibition through inducing Arg1 activity. A, In a blind screen of 12 compounds (3 true hits
in the context of an injured CNS. An ideal
and 9 false positives), only CGNs primed with methoxyvone and daidzein overcame MAG-mediated inhibition of neurite outdrug would be effective following periphgrowth. Daidzein was further characterized due to its superior efficacy. B, Arg1 protein levels are increased by daidzein (20 M) to
the same extent as db-cAMP (2 mM). Arginase activity is required for daidzein-mediated axonal outgrowth on MAG-CHO cells eral administration, either into the sys(CG). CGNs grown on control CHO cells incubated with NOHA (C) exhibit normal neurite outgrowth, whereas CGNs plated on temic circulation (i.e., subcutaneously) or
MAG-CHO cells (D) fail to extend neurites. E, Priming CGNs with daidzein (20 M) for 24 h overcomes MAG inhibition, which was orally. For this to be effective, the drug
reversed by the inclusion of nor-NOHA (10 M), an inhibitor of arginase activity. GI, Neurite outgrowth was quantitated in must also cross the blood brain barrier to
neurons primed with daidzein and then plated on control CHO cells (closed bars), or in neurons grown on MAG-CHO cells (diagonal reach its site of action. To test this, we
bars). P0 CGNs (G), P5P6 DRG neurons (H ), or E18 cortical neurons (I ) treated with daidzein were resistant to inhibition by MAG. implanted daidzein-containing minipumps
These data were repeated in at least three independent experiments, *p 0.001, one-way ANOVA with Tukeys post hoc test.
subcutaneously for 24 h before removing
DRG neurons for plating on MAG-CHO
be effective in diverse cell types in activating pathways to overcells. As the drug must travel through the systemic circulation
come glial inhibition (Cai et al., 1999). To assess whether
when administered by this route, we reasoned that much higher
daidzein, like cAMP, was effective in overcoming MAG inhiconcentrations of daidzein were required than in vitro or for
bition in diverse brain regions, we used DRG neurons (which
local delivery and started with millimolar doses of the drug
have a CNS axon that ascends in the spinal cord) and embryreleased at 0.5 l/h. We found that concentrations of 2 mM
onic cortical neurons. Both DRG (Fig. 2 H) and cortical neuand higher were effective in overcoming MAG inhibition, with
rons (Fig. 2 I) primed with daidzein and subsequently plated
the maximum effect occurring at 12 mM (Fig. 4 F, H ). Toupon MAG-CHO cells were resistant to inhibition of neurite
gether, our results strongly suggest that daidzein is biologically
outgrowth by MAG. Unlike CGNs, the maximal effect of daidactive in vivo, enters the CNS when given peripherally, and
zein occurred from 30 to 40 M in DRG neurons and at 10 M
may be a viable therapeutic agent to promote axonal regenerin cortical neurons, suggesting that the sensitivity to daidzein
ation following CNS injury.
depends on neuronal cell type. Daidzein also rendered growth
cones of adult DRG neurons resistant to the collapse and reDaidzein promotes optic nerve regeneration following
pulsion induced by focal sources of MAG (Fig. 3 A, B,E). As
crush injury
predicted, daidzeins effects are completely abrogated in
To determine whether the beneficial effects of daidzein in the
cultures exposed to DRB (Fig. 3CE), which inhibits 90%
MAG inhibition model translated to improved axonal outgrowth
of new RNA synthesis in these adult DRG cultures
following injury in vivo, we tested daidzein in an optic nerve crush
(Willis et al., 2007). Importantly, these data confirm that daidinjury and regeneration model. The axons of the retinal ganglion

744 J. Neurosci., January 13, 2010 30(2):739 748

neurons, which reside within the CNS,

were mechanically injured 2 mm behind the eye. Immediately following the
crush injury procedure, daidzein (100
M 4 mM) was injected intraocularly,
taking care to not damage the lens. Animals with damaged lenses were not included in the analysis as lens lesions may
also promote recovery (Leon et al., 2000;
Fischer et al., 2001). After 2 weeks, the animals were killed, the injured optic nerves
removed, sectioned, and immunolabeled
for GAP43, a marker for injured/regenerating axons. In vehicle-treated animals,
GAP43 labeling showed that few injured
axons grew beyond the lesion site (Fig. 5A;
asterisk denotes site of injury, arrows denote regenerated axons). In contrast, axons were able to regenerate in animals
receiving daidzein for significant distances beyond the distal margin of the injury (Fig. 5B; DMSO: 436.6 133.8 m vs
daidzein: 2035.2 295.8 m, p 0.05,
Students t test).
As intraocular delivery exposes the retinal ganglion cell bodies directly to daidzein, we wanted to know whether systemic
delivery of daidzein also afforded similar
regenerative enhancement. We used a
postinjury paradigm where daidzein was
delivered subcutaneously with osmotic
minipumps for the 2-week recovery period following the optic nerve crush injury. In agreement with local delivery,
animals receiving subcutaneous daidzein
exhibited longer regenerated axons, extending up to 7 mm beyond the injury site
(Fig. 5C), confirming that daidzein was
effective in promoting axonal regeneration when administered subcutaneously.
Daidzein does not affect cAMP levels or
CREB phosphorylation
The elevation of neuronal cAMP overcomes MAG inhibition by activating PKA,
leading to phosphorylation of CREB at
serine 133, and subsequent recruitment of
the coactivator CBP/p300 to transactivate
a host of genes, including Arg1 (Cai et al.,
2002). To determine whether daidzein affected the cAMP-PKA-CREB pathway, we
measured cAMP levels in cortical neurons
following daidzein treatment and found
that in contrast to db-cAMP, treatment
with daidzein for 2 or 24 h did not increase
intracellular cAMP levels (data not shown
and Fig. 6A, respectively). To further rule
out CREB-mediated induction of Arg1
expression, we treated cortical neurons
with db-cAMP (5 mM) or daidzein (1 40
M; only 20 M is shown), collected protein lysates at 0, 2, 4, 6, and 24 h, and
immunoblotted using an antibody that

Ma et al. Protective Daidzein Overcomes Glial Inhibition

Figure 3. Daidzein prevents growth cone collapse and repulsion from MAG-coated microparticles. Adult DRGs were grown for
12 h in the presence or absence of the RNA polymerase II inhibitor DRB and then treated with vehicle (DMSO) or increasing
concentrations of daidzein for an additional 12 h. A, A representative micrograph of a terminal axon and growth cone, cultured in
the absence of DRB, stimulated with a MAG-Fc-coated microparticle. B, In the absence of DRB, pretreatment with 40 M daidzein
prevents the axon from responding to the MAG stimulus and the axon continues to grow along its trajectory, growing past the
microparticle within 120 min. C, A representative micrograph of a terminal axon and growth cone cultured in the presence of 80 M
DRB, stimulated with a MAG-Fc-coated microparticle. D, Pretreatment with DRB blocks the daidzein effect and the axon turns away
from the MAG stimulus. E, Quantification of axon turning/retraction in response to microparticles loaded with MAG-Fc.
Pretreatment with daidzein at 30 or 40 M attenuates the MAG response. This attenuation is blocked by addition of the
transcriptional inhibitor DRB. These data were repeated in three independent experiments, with at least 20 axons imaged
per experiment, *p 0.001, two-way ANOVA followed by Bonferronis post hoc test.

Figure 4. Daidzein primes in DRG neurons when administered in vivo and crosses the blood brain barrier. Daidzein was
administered as indicated, after which DRG neurons were plated on control or MAG-CHO cells for 24 h, and assayed for neurite
outgrowth by III-tubulin fluorescence immunolabeling. AC, To determine whether in vivo administration of daidzein primes
DRG neurons to overcome MAG inhibition, we infused P28 P30 rats with daidzein intrathecally using osmotic minipumps for 24 h
before removing DRG neurons for culture on CHO cells. A, Neurons from vehicle-infused animals on control CHO cells; B, neurons
from vehicle-infused animals on MAG-CHO cells; C, neurons from animals infused with 40 M daidzein on MAG-CHO cells. DF, To
test whether daidzein crossed the blood brain barrier and was effective in priming DRG neurons when given peripherally,
daidzein was administered to P28 P30 rats through subcutaneously implanted osmotic minipumps for 24 h before removal of
DRG neurons. D, Neurons from vehicle-treated animas on control CHO cells; E, neurons from vehicle-treated animals on MAG-CHO
cells; F, neurons from animals treated with 8 mM daidzein on MAG-CHO cells. G, H, Analysis of neurite lengths from intrathecal
infusion (G) and subcutaneous administration (H ) show that daidzein primes DRG neurons to overcome MAG inhibition when
given in vivo (black bars, neurons plated on control CHO cells; diagonal bars, neurons plated on MAG-CHO cells). The efficacy of
subcutaneous administration suggests that daidzein crosses the blood brain barrier. Data from in vivo priming experiment
represent two animals per condition from two independent experiments, *p 0.01, one-way ANOVA with Tukeys post hoc test.

Ma et al. Protective Daidzein Overcomes Glial Inhibition

J. Neurosci., January 13, 2010 30(2):739 748 745

another estrogen receptor antagonist, although only at high concentrations (data not shown). We next asked whether activation
of estrogen receptors alone was sufficient to induce Arg1 promoter activity. Surprisingly, 17-estradiol was unable to mimic
the effects of daidzein as Arg1 promoter activity remained unchanged (Fig. 6 D). Parallel experiments confirmed that the 17estradiol we used was active, as it could activate luciferase
expression driven by the ERE in HT22 cells (Fig. 6 E).

Figure 5. Daidzein promotes optic nerve regeneration in vivo following an optic nerve crush
injury. The optic nerves of P28 P30 rats were crushed approximate 2 mm behind the eye and
allowed to recover for 2 weeks. Daidzein was administered intraocularly immediately following
the injury, taking care to not damage the lens, or subcutaneously for the 2-week recovery
period. Optic nerves were removed, sectioned, and immunolabeled for GAP43-positive axons
(arrows). A, GAP43-labeled injured optic nerve from an animal that received intraocular vehicle.
B, An injured optic nerve from an animal that received intraocular daidzein. Note the increase in
GAP43-positive axons (arrows) growing beyond the lesion site (asterisk) in daidzein-treated
animals. C, The distance of regeneration beyond the injury site of the three longest axons was
measured in each section from animals treated subcutaneously with vehicle or daidzein. Importantly, these data show that daidzein is effective in an in vivo injury model when given peripherally and after the injury. Data represent mean SEM for 5 animals per group, *p 0.05,
one-way ANOVA with Tukeys post hoc test.

recognizes CREB phosphorylation at serine- 133. With db-cAMP,

maximal phosphorylated CREB (pCREB) levels were reached by
2 h and gradually declined over 24 h. As expected, inhibiting
protein kinase A (PKA) with the pharmacological inhibitor H89
(10 M) prevented the cAMP-induced phosphorylation of CREB
(Fig. 6 B). In contrast, daidzein treatment did not elevate pCREB
levels at any of the time points measured (Fig. 6 B). Thus, we
conclude that daidzein is unlikely to be increasing cAMP levels or
activating PKA to induce Arg1 expression.
Estrogen receptor activity is necessary but insufficient to
activate the Arg1 promoter
Daidzein has been reported to bind and activate estrogen receptors (Choi et al., 2008). To determine whether estrogen receptors
play a role in Arg1 induction, we treated our HT22 Arg1 reporter
cell line with daidzein (20 M) and/or fulvestrant, an estrogen
receptor-specific antagonist. Fulvestrant inhibited both basal and
daidzein-induced Arg1 promoter activity in a dose-dependent
manner (Fig. 6C). We also found similar results with tamoxifen,

Daidzein protects neurons in oxidative models of cell death

Our model posits that agents that can simultaneously recapitulate established benefits of neuroprotective agents and foster regeneration will be superior in promoting recovery after brain and
spinal cord injury compared with established neuroprotective
compounds. Prior studies have reported neuroprotective roles
for daidzein, but the mechanisms for this effect are obscure. As
forced expression of Arg1 is sufficient to protect motor neurons
from trophic factor deprivation-mediated cell death (by inhibiting NO generation and/or generating polyamines), we assessed
whether daidzein could induce Arg1 in motor neurons (Estevez
et al., 2006). Using spinal cord motor neurons purified from
E14.5 rat embryos, we verified that daidzein could induce Arg1
protein expression (Fig. 7A). Motor neurons grown in culture
require the addition of trophic factors such as BDNF. When
plated in the absence of trophic factors, 40% of neurons undergo NO/peroxynitrite-dependent apoptosis, which, as expected, was completely attenuated by daidzein (20 M) (Fig. 7B).
As other oxidants also participate in acute CNS injury, we tested
daidzein in an in vitro model of oxidative injury in cortical neurons. Cell death in this model (embryonic neurons 12 DIV) does
not involve activation of cell surface glutamate receptors (these
receptors are expressed later in vitro (10 21 DIV)) but rather
accrues from the ability of glutamate or its analogs (HCA) to
block uptake of the amino acid cystine into neurons. Cellular
cystine deprivation leads to depletion of the versatile antioxidant
glutathione and NO independent oxidative death (Esch et al.,
1998; Ratan et al., 2002). Treatment of cortical neurons with
HCA for 24 h kills 70% of neurons and this oxidative glutamate
toxicity was significantly inhibited by daidzein (Fig. 7C). Together, these data suggest that daidzein could capture the prior
benefits of neuroprotective agents with the additional benefit of
being able to enhance the ability of preserved neurons to overcome glial inhibition.

Discussion
Agents that increase cAMP such as PDE inhibitors have captured
the attention of those interested in acute and chronic neurological disorders because of their dual ability to foster survival and
enhance regeneration (Gong et al., 2004; DeMarch et al., 2007;
Sasaki et al., 2007; DeMarch et al., 2008; Yang et al., 2008). While
candidate PDE4 inhibitors are moving toward testing in humans
afflicted with CNS injury, side effects such as nausea may limit
their use (Davis et al., 2009). Prior studies by our groups and
others have attributed some of the prosurvival and proregenerative effects of cAMP to the transcriptional upregulation of Arg1
(Cai et al., 2002; Gao et al., 2004).
To identify chemical modulators of Arg1 expression that
could not only be used to develop novel therapeutics, but also to
develop new insight into regulation of the regenerative genetic
program, we screened a well known library of clinically approved
compounds, assembled originally by the NINDS, against a pool
of cell clones expressing an Arg1 promoter-reporter construct. In
completing our screen, we used an unusual methodological fea-

746 J. Neurosci., January 13, 2010 30(2):739 748

Ma et al. Protective Daidzein Overcomes Glial Inhibition

ture that is important to highlight. While

the initial screens to define true positives
(those agents that induced the reporter
and Arg1 protein levels) were performed
in the Ratan Laboratory at Burke, the ability of these agents to overcome glial inhibitors was performed from a set of drugs
containing true positives and false positives by an investigator in the Filbin laboratory at Hunter College. In carrying out
these validation studies, the Hunter team
was blinded to the properties of the compounds. Once the biological analysis was
complete and true positives (e.g., daidzein) were identified, the findings were
replicated (again, with the investigator
blinded to the identity of the compound)
in three other laboratories in three other
institutions (Giger Laboratory at the Uni- Figure 6. Daidzein does not increase cAMP levels or induce CREB phosphorylation but requires estrogen receptor binding to
versity of Michigan, Ratan Laboratory at activate the Arg1 promoter. A, Cortical neurons were treated with daidzein or db-cAMP (5 mM) for 24 h and then assayed for
intracellular cAMP levels. In contrast to db-cAMP, daidzein did not change intracellular cAMP levels. B, Protein lysates from cortical
Burke, and Twiss Laboratory at the A.I.
neuron cultures treated with db-cAMP (5 mM) or daidzein (20 M) were immunoblotted to determine CREB phosphorylation at
duPont Childrens Hospital). Of note, de- Ser-133. A time course shows that db-cAMP maximally induced pCREB levels by 2 h, which gradually declined over 24 h. CREB
spite using distinct sets of cell types and phosphorylation was inhibited by H89 (10 M), a PKA inhibitor, indicating that CREB phosphorylation was PKA dependent. In
assays for these replication experiments, contrast, daidzein did not change pCREB levels at any time point. C, HT22 Arg1 reporter cells were treated with daidzein (20 M)
the concentrationresponse curves were and fulvestrant, an estrogen receptor antagonist. Basal and daidzein-induced Arg1 promoter activity were inhibited by fulvestrant.
virtually identical. Based on our experi- D, E, 17-Estradiol alone (D) is insufficient to activate the Arg1 promoter at concentrations that activate the ERE in HT22 cells (E).
ence, we would propose that translational Experiments were repeated at least three times with identical results. *p 0.001, two-way ANOVA with Bonferronis post hoc test.
efforts to identify novel drugs include a
similar replication strategy in advance of
publication (Steward et al., 2008).
Two critical pieces of evidence suggest
that daidzein acts via the transcriptional
upregulation of Arg1 to mediate the biological effects described herein. First, the
canonical Arg1 inhibitor nor-NOHA
completely abrogated the ability of daidzein to overcome inhibition of neurite
growth by MAG, whereas nor-NOHA
Figure 7. Daidzein protects neurons in two models of oxidative injury. Protein lysates from spinal cord motor neurons treated
alone had no effect (Fig. 2G). Second, the
with daidzein (0 20 M) for 24 h were immunoblotted to determine Arg1 protein levels. A, Representative Western blot of Arg1
ability of daidzein to render growth cones levels in motor neurons following daidzein treatment. B, Motor neurons were plated in the presence (closed bars) or absence
resistant to collapse by MAG was depen- (diagonal bars) of BDNF. Omission of BDNF causes peroxynitrite-dependent motor neuron apoptosis, which was blocked by
dent on transcription (Fig. 3). Future daidzein (20 M). C, Cortical neurons were treated with (diagonal bars) or without (closed bar) HCA, a compound that depletes
studies using conditional knock-outs of intraneuronal glutathione and increases oxidative stress. Neurons treated with HCA undergo apoptosis, which is also blocked by
Arg1 will examine the necessity of Arg1 daidzein (20 M). Data represent mean SEM of 4 6 independent experiments, *p 0.05 one-way ANOVA with Newman
Keuls post hoc test.
for daidzeins effects in vivo.
Given that at least some of the effects of
zein on estrogen receptors (Zhao et al., 2002; Wang et al., 2008;
daidzein are mediated via the transcriptional induction of Arg1,
Schreihofer and Redmond, 2009). While the induction of Arg1 by
an important unanswered question is how daidzein executes
daidzein was estrogen receptor dependent, 17-estradiol alone
these functions. Our data suggests that Arg1 is induced by cAMPdid not activate the Arg1 promoter (Fig. 6C,D), indicating that
and CREB-independent mechanisms as intracellular cAMP levels
although estrogenic activity is required, daidzein possesses addiand CREB phosphorylation were unchanged by daidzein treattional activities that contribute to Arg1 induction. Accordingly,
ment (Fig. 6 A, B), indicating that daidzein is not activating adegenistein, which is more estrogenic than daidzein, also failed to
nylate cyclase, inhibiting PDEs, or engaging other CREBinduce Arg1 protein levels. Future studies will exploit the chemdependent pathways to induce Arg1 transcription. Daidzein has
ical similarity between daidzein and genistein to identify the as
been evaluated in humans as an estrogen supplement and alyet unestablished activity of daidzein that endows regenerative
though these trials have been disappointing, they highlight an
capacities to an estrogen agonist. However, we cannot discern at
important biological property of isoflavones as agonists of the
this time whether the inability of genistein (which differs in one
estrogen receptors, which are known transcriptional activators
hydroxyl group from daidzein) to activate Arg1 relates to a pos(Cassidy et al., 2006; Heldring et al., 2007). Daidzein has been
itive activity of daidzein or a negative activity of genistein that is
reported to be neuroprotective to CNS neurons in glutamate
not present in daidzein.
excitotoxicity and oxygen/glucose deprivation models and to
In contrast to db-cAMP, neurons required priming with daidpromote axonal outgrowth of hippocampal neurons on permiszein to overcome MAG inhibition in vitro. This difference reflects
sive substrates; these effects were attributed to the activity of daid-

Ma et al. Protective Daidzein Overcomes Glial Inhibition

the biphasic response of neurons to cAMP in overcoming MAG

inhibition, which consists of an immediate PKA-dependent and
transcription-independent mechanism followed by a delayed
PKA-independent activation of transcription (Qiu et al., 2002).
As daidzein does not activate PKA, there is, as expected, no observed transcription independent phase to overcome glial inhibition. Thus priming with daidzein allows neurons to transcribe
and accumulate effector proteins, such as Arg1, to resist glial
inhibitors. Interestingly, daidzein did not increase axonal growth
on permissive substrates (Fig. 2 H, I ), suggesting that the primary
effect of daidzein may be to overcome external inhibitory cues
(such as MAG). Additional factors that increase baseline growth,
such as neurotrophins, may augment the effects of daidzein on
neurons grown in an inhibitory environment; these combinational studies are ongoing.
Daidzein was effective in promoting axonal regeneration in an
optic nerve crush model of CNS injury without lens damage in
vivo. Though some optic nerve axons grew significant distances
beyond the injury site, relatively few axons regenerated (Fig. 5),
which is reminiscent of enhanced optic nerve regeneration from
Rho inactivation, ROCK inhibition, and intraocular spermidine
(Lehmann et al., 1999; Bertrand et al., 2005; Lingor et al., 2007;
Deng et al., 2009). As such, we used the average length of the three
longest axons as a measure of regeneration to highlight this property of daidzein. Of note, our lowest dose of subcutaneous daidzein was most effective, suggesting an U-shaped doseresponse
relationship and that our in vivo dosing protocols may require
further optimization. Future studies will assess the proper dose/
duration of daidzein treatment to ensure that RGCs are exposed
to the optimal amount of daidzein to see whether we can achieve
the robust regeneration observed after manipulations such as
inducing intraocular inflammation, oncomodulin injection, or
genetic PTEN deletion (Yin et al., 2003, 2006; Park et al., 2008).
In this CNS injury model, puncture of the lens alone promotes
significant regeneration, which has been attributed to a change in
growth state of RGNs due to factors released by invading macrophages (Leon et al., 2000; Fischer et al., 2001; Yin et al., 2003,
2006). Lens injury also increases the efficacy of certain therapeutic manipulations (Fischer et al., 2004a,b). Whether lens puncture is required to enhance the number regenerating axons with
daidzein is yet to be determined, though this possibility would
limit its value for monotherapy. Interestingly, lens injury induces
many gene expression changes, including the upregulation of
Arg1, suggesting that daidzein may activate a similar transcriptional program (Fischer et al., 2004b). The mechanistic and biological congruence between lens injury and daidzein treatment
may set the table for cotreatment with other therapeutic agents
such as ROCK inhibitors.
Excitingly, daidzein was effective in vivo when given subcutaneously, indicating that it crossed the blood brain barrier to
enter the site of injury in the CNS. In agreement with our observation, one study detected daidzein in the brain of rats receiving
an enteral mixture of soy proteins (Gu et al., 2005). However, we
have not ruled out the possibility of a peripheral action of
daidzein, such as a global increase in polyamine levels. Daidzein administered continuously for 2 weeks was more effective in
promoting axon regeneration than an intraocular bolus injection
following injury, suggesting a benefit for chronic application. The
finding also suggests that regenerating axons may remain responsive to daidzein for extended periods postinjury. Future studies
will determine the precise therapeutic window.
While daidzein is far more potent than analogs of cAMP in
inducing Arg1 expression, the potency of daidzein for promoting

J. Neurosci., January 13, 2010 30(2):739 748 747

survival and regeneration presented in this manuscript (Figs. 1C,

2 B,GI, 3E) is unlikely to be sufficient for the compound to be
effective as a drug in humans. With oral administration, daidzein
and its metabolites (e.g., equol) rarely reach concentrations 1
M (Nielsen and Williamson, 2007). However, even in the absence of a clear target, natural and synthetic analogs of daidzein
are being developed that may enhance potency and efficacy to
allow oral administration.
Despite these caveats, preliminary observations of subcutaneous daidzein initiated early following stroke in an animal model
indicate that the unmodified daidzein can enhance functional
recovery when behavior is analyzed 1 month after the ictus (Cho
et al., unpublished observations). These promising observations
suggest that human trials of daidzein given as soon as possible
after the stroke or spinal cord injury and continued for weeks to
months afterward may not be far off. The success of daidzein in
more complex animal models of human disease could also provide affirmation of our Arg1promoter-reporter screen for identifying a new generation of neuroprotective agents with extant
regenerative properties.

References
Baptiste DC, Fehlings MG (2007) Update on the treatment of spinal cord
injury. Prog Brain Res 161:217233.
Bertrand J, Winton MJ, Rodriguez-Hernandez N, Campenot RB, McKerracher L (2005) Application of Rho antagonist to neuronal cell bodies
promotes neurite growth in compartmented cultures and regeneration of
retinal ganglion cell axons in the optic nerve of adult rats. J Neurosci
25:11131121.
Block F, Tondar A, Schmidt W, Schwarz M (1997) Delayed treatment with
rolipram protects against neuronal damage following global ischemia in
rats. Neuroreport 8:3829 3832.
Cafferty WB, Strittmatter SM (2006) The Nogo-Nogo receptor pathway
limits a spectrum of adult CNS axonal growth. J Neurosci 26:
1224212250.
Cai D, Shen Y, De Bellard M, Tang S, Filbin MT (1999) Prior exposure to
neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron 22:89 101.
Cai D, Deng K, Mellado W, Lee J, Ratan RR, Filbin MT (2002) Arginase I
and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro. Neuron 35:711719.
Cassidy A, Albertazzi P, Lise Nielsen I, Hall W, Williamson G, Tetens I, Atkins
S, Cross H, Manios Y, Wolk A, Steiner C, Branca F (2006) Critical review
of health effects of soyabean phyto-oestrogens in post-menopausal
women. Proc Nutr Soc 65:76 92.
Choi SY, Ha TY, Ahn JY, Kim SR, Kang KS, Hwang IK, Kim S (2008) Estrogenic activities of isoflavones and flavones and their structure-activity
relationships. Planta Med 74:2532.
Davis TG, Peterson JJ, Kou JP, Capper-Spudich EA, Ball D, Nials AT, Wiseman J,
Solanke YE, Lucas FS, Williamson RA, Ferrari L, Wren P, Knowles RG,
Barnette MS, Podolin PL (2009) The identification of a novel phosphodiesterase 4 inhibitor, 1-ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2 H-pyran-4-yl)-1 H-pyrazolo[3,4-b]
pyridin-4-amine (EPPA-1), with improved therapeutic index using pica
feeding in rats as a measure of emetogenicity. J Pharmacol Exp Ther
330:922931.
Dawson VL, Dawson TM, London ED, Bredt DS, Snyder SH (1991) Nitric
oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc
Natl Acad Sci U S A 88:6368 6371.
DeMarch Z, Giampa` C, Patassini S, Martorana A, Bernardi G, Fusco FR
(2007) Beneficial effects of rolipram in a quinolinic acid model of striatal
excitotoxicity. Neurobiol Dis 25:266 273.
DeMarch Z, Giampa` C, Patassini S, Bernardi G, Fusco FR (2008) Beneficial
effects of rolipram in the R6/2 mouse model of Huntingtons disease.
Neurobiol Dis 30:375387.
Deng K, He H, Qiu J, Lorber B, Bryson JB, Filbin MT (2009) Increased
synthesis of spermidine as a result of upregulation of arginase I promotes
axonal regeneration in culture and in vivo. J Neurosci 29:95459552.
Esch F, Lin KI, Hills A, Zaman K, Baraban JM, Chatterjee S, Rubin L, Ash DE,

748 J. Neurosci., January 13, 2010 30(2):739 748

Ratan RR (1998) Purification of a multipotent antideath activity from
bovine liver and its identification as arginase: nitric oxide-independent
inhibition of neuronal apoptosis. J Neurosci 18:4083 4095.
Estevez AG, Sahawneh MA, Lange PS, Bae N, Egea M, Ratan RR (2006)
Arginase 1 regulation of nitric oxide production is key to survival of
trophic factor-deprived motor neurons. J Neurosci 26:8512 8516.
Filbin MT (2003) Myelin-associated inhibitors of axonal regeneration in the
adult mammalian CNS. Nat Rev Neurosci 4:703713.
Fischer D, Heiduschka P, Thanos S (2001) Lens-injury-stimulated axonal
regeneration throughout the optic pathway of adult rats. Exp Neurol
172:257272.
Fischer D, He Z, Benowitz LI (2004a) Counteracting the Nogo receptor
enhances optic nerve regeneration if retinal ganglion cells are in an active
growth state. J Neurosci 24:1646 1651.
Fischer D, Petkova V, Thanos S, Benowitz LI (2004b) Switching mature
retinal ganglion cells to a robust growth state in vivo: gene expression and
synergy with RhoA inactivation. J Neurosci 24:8726 8740.
Gao Y, Deng K, Hou J, Bryson JB, Barco A, Nikulina E, Spencer T, Mellado W,
Kandel ER, Filbin MT (2004) Activated CREB is sufficient to overcome
inhibitors in myelin and promote spinal axon regeneration in vivo. Neuron 44:609 621.
Gong B, Vitolo OV, Trinchese F, Liu S, Shelanski M, Arancio O (2004)
Persistent improvement in synaptic and cognitive functions in an Alzheimer mouse model after rolipram treatment. J Clin Invest 114:1624 1634.
Gray MJ, Poljakovic M, Kepka-Lenhart D, Morris SM Jr (2005) Induction
of arginase I transcription by IL-4 requires a composite DNA response
element for STAT6 and C/EBPbeta. Gene 353:98 106.
Gu L, Laly M, Chang HC, Prior RL, Fang N, Ronis MJ, Badger TM (2005)
Isoflavone conjugates are underestimated in tissues using enzymatic hydrolysis. J Agric Food Chem 53:6858 6863.
Hall JM, McDonnell DP (1999) The estrogen receptor beta-isoform (ERbeta)
of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566 5578.
Hannila SS, Filbin MT (2008) The role of cyclic AMP signaling in promoting axonal regeneration after spinal cord injury. Exp Neurol 209:321332.
Heemskerk J, Tobin AJ, Bain LJ (2002) Teaching old drugs new tricks. Meeting of the Neurodegeneration Drug Screening Consortium, 7 8 April
2002, Washington, DC. Trends Neurosci 25:494 496.
Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, Tujague
M, Strom A, Treuter E, Warner M, Gustafsson JA (2007) Estrogen receptors: how do they signal and what are their targets. Physiol Rev
87:905931.
Kato H, Araki T, Itoyama Y, Kogure K (1995) Rolipram, a cyclic AMPselective phosphodiesterase inhibitor, reduces neuronal damage following cerebral ischemia in the gerbil. Eur J Pharmacol 272:107110.
Lange PS, Langley B, Lu P, Ratan RR (2004) Novel roles for arginase in cell
survival, regeneration, and translation in the central nervous system.
J Nutr 134:2812S2817S; discussion 2812S2817S.
Lehmann M, Fournier A, Selles-Navarro I, Dergham P, Sebok A, Leclerc N,
Tigyi G, McKerracher L (1999) Inactivation of Rho signaling pathway
promotes CNS axon regeneration. J Neurosci 19:75377547.
Leon S, Yin Y, Nguyen J, Irwin N, Benowitz LI (2000) Lens injury stimulates
axon regeneration in the mature rat optic nerve. J Neurosci 20:4615
4626.
Lingor P, Teusch N, Schwarz K, Mueller R, Mack H, Bahr M, Mueller BK
(2007) Inhibition of Rho kinase (ROCK) increases neurite outgrowth on
chondroitin sulphate proteoglycan in vitro and axonal regeneration in the
adult optic nerve in vivo. J Neurochem 103:181189.
Lu P, Yang H, Jones LL, Filbin MT, Tuszynski MH (2004) Combinatorial
therapy with neurotrophins and cAMP promotes axonal regeneration
beyond sites of spinal cord injury. J Neurosci 24:6402 6409.
Mehta NR, Lopez PH, Vyas AA, Schnaar RL (2007) Gangliosides and Nogo
receptors independently mediate myelin-associated glycoprotein inhibition of neurite outgrowth in different nerve cells. J Biol Chem 282:27875
27886.
Mukhopadhyay G, Doherty P, Walsh FS, Crocker PR, Filbin MT (1994) A

Ma et al. Protective Daidzein Overcomes Glial Inhibition

novel role for myelin-associated glycoprotein as an inhibitor of axonal
regeneration. Neuron 13:757767.
Neumann S, Bradke F, Tessier-Lavigne M, Basbaum AI (2002) Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation. Neuron 34:885 893.
Nielsen IL, Williamson G (2007) Review of the factors affecting bioavailability of soy isoflavones in humans. Nutr Cancer 57:110.
Nikulina E, Tidwell JL, Dai HN, Bregman BS, Filbin MT (2004) The phosphodiesterase inhibitor rolipram delivered after a spinal cord lesion promotes axonal regeneration and functional recovery. Proc Natl Acad Sci
U S A 101:8786 8790.
Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, Xu B, Connolly L, Kramvis
I, Sahin M, He Z (2008) Promoting axon regeneration in the adult CNS
by modulation of the PTEN/mTOR pathway. Science 322:963966.
Pearse DD, Pereira FC, Marcillo AE, Bates ML, Berrocal YA, Filbin MT,
Bunge MB (2004) cAMP and Schwann cells promote axonal growth and
functional recovery after spinal cord injury. Nat Med 10:610 616.
Qiu J, Cai D, Dai H, McAtee M, Hoffman PN, Bregman BS, Filbin MT (2002)
Spinal axon regeneration induced by elevation of cyclic AMP. Neuron
34:895903.
Ratan RR, Noble M (2009) Novel multi-modal strategies to promote brain
and spinal cord injury recovery. Stroke 40:S130 132.
Ratan RR, Ryu H, Lee J, Mwidau A, Neve RL (2002) In vitro model of
oxidative stress in cortical neurons. Methods Enzymol 352:183190.
Rossignol S, Schwab M, Schwartz M, Fehlings MG (2007) Spinal cord injury: time to move? J Neurosci 27:1178211792.
Sasaki T, Kitagawa K, Omura-Matsuoka E, Todo K, Terasaki Y, Sugiura S,
Hatazawa J, Yagita Y, Hori M (2007) The phosphodiesterase inhibitor
rolipram promotes survival of newborn hippocampal neurons after ischemia. Stroke 38:15971605.
Savitz SI, Fisher M (2007) Future of neuroprotection for acute stroke: in the
aftermath of the SAINT trials. Ann Neurol 61:396 402.
Schreihofer DA, Redmond L (2009) Soy phytoestrogens are neuroprotective against stroke-like injury in vitro. Neuroscience 158:602 609.
Spina D (2008) PDE4 inhibitors: current status. Br J Pharmacol 155:
308 315.
Steward O, Sharp K, Yee KM, Hofstadter M (2008) A re-assessment of the
effects of a Nogo-66 receptor antagonist on regenerative growth of axons
and locomotor recovery after spinal cord injury in mice. Exp Neurol
209:446 468.
Twiss JL, Smith DS, Chang B, Shooter EM (2000) Translational control of
ribosomal protein L4 mRNA is required for rapid neurite regeneration.
Neurobiol Dis 7:416 428.
Venkatesh K, Chivatakarn O, Sheu SS, Giger RJ (2007) Molecular dissection
of the myelin-associated glycoprotein receptor complex reveals cell typespecific mechanisms for neurite outgrowth inhibition. J Cell Biol 177:
393399.
Wang P, Jeng CJ, Chien CL, Wang SM (2008) Signaling mechanisms of
daidzein-induced axonal outgrowth in hippocampal neurons. Biochem
Biophys Res Commun 366:393 400.
Willis DE, van Niekerk EA, Sasaki Y, Mesngon M, Merianda TT, Williams
GG, Kendall M, Smith DS, Bassell GJ, Twiss JL (2007) Extracellular
stimuli specifically regulate localized levels of individual neuronal mRNAs. J Cell Biol 178:965980.
Yang L, Calingasan NY, Lorenzo BJ, Beal MF (2008) Attenuation of MPTP
neurotoxicity by rolipram, a specific inhibitor of phosphodiesterase IV.
Exp Neurol 211:311314.
Yin Y, Cui Q, Li Y, Irwin N, Fischer D, Harvey AR, Benowitz LI (2003)
Macrophage-derived factors stimulate optic nerve regeneration. J Neurosci 23:2284 2293.
Yin Y, Henzl MT, Lorber B, Nakazawa T, Thomas TT, Jiang F, Langer R,
Benowitz LI (2006) Oncomodulin is a macrophage-derived signal for
axon regeneration in retinal ganglion cells. Nat Neurosci 9:843 852.
Zhao L, Chen Q, Diaz Brinton R (2002) Neuroprotective and neurotrophic
efficacy of phytoestrogens in cultured hippocampal neurons. Exp Biol
Med (Maywood) 227:509 519.